A synthetic method of oligopeptide-1

Abstract

The invention discloses a method for synthesizing oligopeptide 1, which belongs to the technical field of polypeptide synthesis, and comprises the following steps: synthesizing H-Gly-His-OH, synthesizing Boc-Gly-His(Boc)-OH, synthesizing Boc-Gly-His(Boc)-Lys(Boc)-OH, synthesizing H-Gly-His-Lys-OH, and obtaining oligopeptide 1. The invention has the beneficial effects that each step of the method is simple and fast to synthesize, and high-quality products can be obtained. Through four-step reaction, dipeptide can be quickly synthesized, two protections are applied in one step, all protections are removed in one step, and the final product can be recrystallized to obtain a qualified product, with a short production cycle and low production cost.

Description

Technical field:

[0001] The invention belongs to the technical field of polypeptide synthesis, and more specifically relates to a method for synthesizing oligopeptide-1. Background technology:

[0002] Oligopeptide-1 is a matrikine messenger peptide that acts on the dermis. It is an amino acid sequence in the ECM protein and can be released by the protease at the wound site. The released molecules can activate and regulate the skin repair process and effectively promote the synthesis of extracellular matrix such as type I and III collagen, elastin, structural glycoproteins such as laminin and fibronectin. At the same time, oligopeptide-1 can reduce the secretion of IL-6 induced by TNF-alpha, reduce the impact of inflammation on the skin, promote wound recovery and tissue renewal and reconstruction, strengthen the dermis, make the skin thicker and firmer, relieve wrinkles, and have a stronger ability to resist ultraviolet radiation. Oligopeptide-1 is the smallest molecular weight beauty tripeptide besides carnosine. Due to its small molecular weight, it is easily absorbed and utilized by human skin and has higher skin permeability than polypeptides. In addition, oligopeptide-1 also has many characteristics such as non-toxicity, multiple active functions, specific biochemical and physiological mechanisms, and homology with body substances, which can essentially improve a series of skin problems. In general, oligopeptide-1 has powerful effects such as improving wrinkles, skin color, acne, and repairing skin damage.

[0003] Currently, there are very few literature reports on the synthesis of oligopeptide-1, and there is no published invention patent. Solid-phase synthesis uses a lot of solvents, a lot of waste liquid, expensive raw materials, low yield, and high cost; conventional liquid-phase synthesis requires protection and deprotection of each amino acid, with long steps, long cycles, many side reactions, and many impurities. Summary of the invention:

[0004] In order to solve the above problems and overcome the shortcomings of the prior art, the present invention provides a method for synthesizing oligopeptide-1 that can effectively solve the problems of high cost and long cycle.

[0005] The specific technical solution of the present invention to solve the above technical problem is as follows: A method for synthesizing oligopeptide-1, comprising the following steps:

[0006] Step 1: Synthesis of H-Gly-His-OH

[0007]

[0008] Glycine and a reaction solvent were added to a container, and triphosgene was added under an ice bath. After reacting at an internal temperature of 25±2°C for 3 hours, the solvent was concentrated, and the residue was added to methyl tert-butyl ether and filtered to obtain a yellow solid 1. Water and L-histidine were added to the container, and the pH was adjusted to 10.2±0.2 with a 30% sodium hydroxide solution. The solid 1 was added at a temperature of 10-15°C, and the pH of the feed solution was controlled to 10.2±0.2 with a sodium hydroxide solution. After the addition was completed, the reaction was continued at an external temperature of 25±2°C for 1 hour. The pH was adjusted to 7.0-7.5, the water was concentrated, ethanol was added, stirred and crystallized for 2 hours, and then filtered. The filter cake was dried in vacuo at 45±2°C to obtain H-Gly-His-OH.

[0009] The molar ratio of glycine: triphosgene: histidine is 1: (1.05-1.2): (0.95-1); the mass ratio of glycine: reaction solvent: methyl tert-butyl ether: water: ethanol is 1: (3-5): (5-6): 5: (13-15);

[0010] Step 2: Synthesis of Boc-Gly-His(Boc)-OH

[0011]

[0012] Add reaction solvent, H-Gly-His-OH and Boc anhydride into the container, stir to dissolve, maintain the internal temperature at 5-10 degrees, add triethylamine dropwise, and after the addition is complete, react at an internal temperature of 45±2°C for 3 hours, and concentrate to dryness at 55±2°C to obtain Boc-Gly-His(Boc)-OH.

[0013] The molar ratio of H-Gly-His-OH: Boc anhydride: triethylamine is 1: (2.2-2.4): (2.2-2.4); the mass ratio of H-Gly-His-OH: reaction solvent is 1: (5-6);

[0014] Step 3: Synthesis of Boc-Gly-His(Boc)-Lys(Boc)-OH

[0015]

[0016] Add solvent, Oxyma, Boc-Gly-His(Boc)-OH to the container, stir to dissolve, maintain the internal temperature at 5-10℃, and drop the mixed solution of DCC; after the drop is complete, react at the internal temperature of 25±2℃ for 3h; add water, H--Lys(Boc)-OH, and a mixed solution of sodium bicarbonate and keep warm for 2h; after the reaction is completed by HPLC detection, filter, and concentrate the filtrate at an external temperature of 45±2℃. Adjust the pH to 2.2±0.2 and extract once with ethyl acetate. Wash the ethyl acetate phase with acid water at pH 2.2±0.2 three times. Wash the washed ethyl acetate phase with saturated brine once, dry with anhydrous magnesium sulfate, and filter. Concentrate the filtrate at an external temperature of 45±2℃ until no more is obtained to obtain a light yellow oil. Add acetone to the bottle containing the oil, stir at an external temperature of 60±2°C until it dissolves, then cool to room temperature naturally to precipitate a solid, filter it with suction, and dry the filter cake at an external temperature of 40±2°C to obtain Boc-Gly-His(Boc)-Lys(Boc)-OH.

[0017] The molar ratio of Boc-Gly-His(Boc)-OH:Oxyma:DCC:H--Lys(Boc)-OH:sodium bicarbonate is 1:(1.05-1.2):(1.05-1.2):(0.95-1.0):(1.05-1.2); the mass ratio of Boc-Gly-His(Boc)-OH:reaction solvent:water:ethyl acetate:acid water:saturated brine:anhydrous magnesium sulfate:acetone is 1:(5-6);2;(3-4);2;2;0.15;(2-3);

[0018] Step 4: Synthesis of H-Gly-His-Lys-OH

[0019]

[0020] Add concentrated hydrochloric acid and Boc-Gly-His(Boc)-Lys(Boc)-OH to the container, stir to dissolve, and then raise the internal temperature to 90±2℃ for 6h. After the reaction is completed by HPLC, lower the internal temperature to 25±2℃, add THF to the reaction solution, grow crystals for 3h, and then filter to obtain a white solid; the solid is then stirred and dissolved by adding methanol, and the solvent is added dropwise until the feed liquid is turbid. Maintain the internal temperature at 0-5 degrees, crystallize for 10h, filter, and dry the filter cake at an external temperature of 40±2℃ to obtain oligopeptide-1.

[0021] The mass ratio of Boc-Gly-His(Boc)-Lys(Boc)-OH:concentrated hydrochloric acid:tetrahydrofuran:methanol:crystallization solvent is 1:(1.1-1.2);(4.5-5.5);(1.8-2.2);(3-4);

[0022] The reaction solvents used in each step are THF and acetonitrile; the crystallization solvent in step 4 is diethyl ether, isopropyl ether, and tert-methyl ether.

[0023] The beneficial effects of the present invention are as follows: each step of the method is simple and fast in synthesis, and a high-quality product can be obtained. Through four-step reaction, a dipeptide can be quickly synthesized, two protections are applied in one step, all protections are removed in one step, and a qualified product can be obtained by recrystallization of the final product, with a short production cycle and low production cost. Description of the drawings:

[0024] Attached Figure 1 It is a schematic diagram of the purity spectrum of oligopeptide-1 in Example 1 of the present invention;

[0025] Attached Figure 2 It is a schematic diagram of the purity spectrum of oligopeptide-1 in Example 2 of the present invention;

[0026] Attached Figure 3 It is a schematic diagram of the purity spectrum of oligopeptide-1 in Example 3 of the present invention;

[0027] Attached Figure 4 It is a schematic diagram of the purity spectrum of oligopeptide-1 in comparative example 1 of the present invention;

[0028] Attached Figure 5 It is a schematic diagram of the purity spectrum of oligopeptide-1 in comparative example 2 of the present invention;

[0029] Attached Figure 6 It is a schematic diagram of the purity spectrum of oligopeptide-1 in comparative example 3 of the present invention. Specific implementation method:

[0030] In the description of the present invention, specific details are only for a full understanding of the embodiments of the present invention, but those skilled in the art should know that the implementation of the present invention is not limited to these details. In addition, well-known structures and functions are not described or shown in detail to avoid blurring the key points of the embodiments of the present invention. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0031] Specific implementation of the present invention:

[0032] In order to better understand the present invention, a specific embodiment is used for illustration. It is worth emphasizing that the effect of this embodiment is not substantially different from the various embodiments within the protection scope of the present invention, including the respective reagents and the content ratio of the reagents, and all of them can achieve the effects described in the present invention and solve the above-mentioned problems. Other combinations are not described here.

[0033] Embodiment 1:

[0034] Step 1: Synthesis of H-Gly-His-OH

[0035] 1 kg THF and 200 g H-Gly-OH were added to a 5L three-necked flask, and 948.69 g triphosgene was added under an ice bath. After the addition, the internal temperature was 25 degrees and the reaction was continued for 3 hours. The THF was concentrated, and the residue was added to 1.2 kg methyl tert-butyl ether and filtered to obtain a yellow solid 1. 1 kg water and 413.38 g L-histidine were added to a 5L three-necked flask, and the pH was adjusted to 10.2 with a 30% sodium hydroxide solution. The solid 1 was added at a temperature of 10-15 ° C, and the pH of the feed solution was controlled to 10.0-10.4 with a sodium hydroxide solution. After the addition was completed, the external temperature was 25 degrees and the reaction was continued for 1 hour; the pH was adjusted to 7.0, the water was concentrated, 3 kg ethanol was added, and the mixture was stirred and crystallized for 2 hours, then filtered, and the filter cake was vacuum dried at 45 degrees to obtain 536.6 g of an off-white solid with a yield of 94.9%.

[0036] Step 2: Synthesis of Boc-Gly-His(Boc)-OH

[0037] In a 10L three-necked flask, add 3.22kg THF, 536.6g H-Gly-His-OH, and 1.32kg Boc anhydride, stir until dissolved, maintain the internal temperature at 5-10 degrees, dropwise add 614.11g triethylamine, after the dropwise addition is complete, react at an internal temperature of 45 degrees for 3h, and concentrate to dryness at 55 degrees to obtain 988g of light yellow oil. Yield: 95.0%.

[0038] Step 3: Synthesis of Boc-Gly-His(Boc)-Lys(Boc)-OH

[0039] In a 10L three-necked flask, add 4700g THF, 408.52g Oxyma, 988g Boc-Gly-His(Boc)-OH, stir and dissolve, keep the internal temperature at 5-10 degrees, add DCC THF solution (593.12g DCC is dissolved in 1200g THF); after the dripping is completed, react at an internal temperature of 25 degrees for 3 hours; add 1976g water, 590.02g H--Lys(Boc)-OH, and a mixed solution of 241.67g sodium bicarbonate and keep warm for 2 hours. After the reaction is completed by HPLC detection, filter, and concentrate the filtrate at an external temperature of 45 degrees. Adjust the pH to 2.0 and extract once with 3952g ethyl acetate. Wash the ethyl acetate phase with acid water at pH 2.0 three times, 1976g each time. Wash the washed ethyl acetate phase with 1976g saturated sodium chloride once, dry with 148.2g anhydrous magnesium sulfate, and filter. The filtrate was concentrated at an external temperature of 45 degrees to obtain a light yellow oil. 2964g of acetone was added to the bottle containing the oil, and the temperature was 60 degrees to stir and dissolve, and then the temperature was naturally cooled to room temperature to precipitate solids, which were filtered by suction and the filter cake was dried at an external temperature of 40 degrees to obtain 1.45kg of off-white solids with a yield of 94.77%.

[0040] Step 4: Synthesis of H-Gly-His-Lys-OH

[0041] 5L three-necked flask, add 1.74kg concentrated hydrochloric acid, 1.45kg Boc-Gly-His(Boc)-Lys(Boc)-OH, stir to dissolve, raise the internal temperature to 90 degrees and react for 6 hours. After the reaction is completed by HPLC detection, lower the internal temperature to 25℃, add 7975g THF to the reaction solution, grow crystals for 3 hours, and then filter to obtain a white solid; the solid is then added with 3190g methanol to stir and dissolve, and 5800g ether is added dropwise until the feed liquid is turbid. Maintain the internal temperature at 0-5 degrees, crystallize for 10 hours, filter with suction, and dry the filter cake at an external temperature of 40 degrees to obtain 723g white solid, yield: 94.0%.

[0042] As a result, the purity of the obtained product was 98.77% and the total yield was 80.3%.

[0043] Embodiment 2:

[0044] Step 1: Synthesis of H-Gly-His-OH

[0045] 800g acetonitrile and 200g H-Gly-OH were added to a 5L three-necked flask, and 869.63g triphosgene was added under an ice bath. After the addition, the internal temperature was 25 degrees and the reaction was continued for 3h. The acetonitrile was concentrated, and the residue was added to 1.1kg methyl tert-butyl ether and filtered to obtain a yellow solid 1; 1kg water and 405.11g L-histidine were added to a 5L three-necked flask, and the pH was adjusted to 10.2 with 30% sodium hydroxide solution. The solid 1 was added at a temperature of 10-15°C, and the pH of the feed liquid was controlled to 10.0-10.4 with sodium hydroxide solution. After the addition was completed, the external temperature was 25 degrees and the reaction was continued for 1h; the pH was adjusted to 7.0, the water was concentrated, 2.8kg ethanol was added, stirred and crystallized for 2h, and then filtered. The filter cake was vacuum dried at 45 degrees to obtain 525.8g off-white solid, with a yield of 94.9%.

[0046] Step 2: Synthesis of Boc-Gly-His(Boc)-OH

[0047] In a 10L three-necked flask, add 2.89kg acetonitrile, 525.8g H-Gly-His-OH, and 1.24kg Boc anhydride, stir until dissolved, maintain the internal temperature at 5-10 degrees, dropwise add 576.68g triethylamine, after the dropwise addition is complete, react at an internal temperature of 45 degrees for 3 hours, and concentrate to dryness at 55 degrees to obtain 971g light yellow oil. Yield: 95.2%.

[0048] Step 3: Synthesis of Boc-Gly-His(Boc)-Lys(Boc)-OH

[0049] In a 10L three-necked flask, add 4250g acetonitrile, 368.03g Oxyma, 971g Boc-Gly-His(Boc)-OH, stir and dissolve, keep the internal temperature at 5-10 degrees, and drop the acetonitrile solution of DCC (534.34g DCC is dissolved in 1100g acetonitrile); after the dripping is completed, react at an internal temperature of 25 degrees for 3 hours; add 1942g water, 568.27g H--Lys(Boc)-OH, and a mixed solution of 217.14g sodium bicarbonate and keep warm for 2 hours. After the reaction is completed by HPLC detection, filter, and concentrate the filtrate at an external temperature of 45 degrees. Adjust the pH to 2.0 and extract once with 3400g ethyl acetate. Wash the ethyl acetate phase with acid water at pH 2.0 three times, 1942g each time. Wash the washed ethyl acetate phase with 1942g saturated sodium chloride once, dry with 146g anhydrous magnesium sulfate, and filter. The filtrate was concentrated at an external temperature of 45 degrees to obtain a light yellow oil. 2430g of acetone was added to the bottle containing the oil, and the temperature was 60 degrees to stir and dissolve, and then the temperature was naturally cooled to room temperature to precipitate solids, which were filtered by suction and the filter cake was dried at an external temperature of 40 degrees to obtain 1.406kg of off-white solids with a yield of 95.0%.

[0050] Step 4: Synthesis of H-Gly-His-Lys-OH

[0051] 5L three-necked flask, add 1.48kg concentrated hydrochloric acid, 1.406kg Boc-Gly-His(Boc)-Lys(Boc)-OH, stir to dissolve, raise the internal temperature to 90 degrees and react for 6 hours. After the reaction is completed by HPLC detection, lower the internal temperature to 25℃, add 7030g acetonitrile to the reaction solution, grow crystals for 3 hours, and then filter to obtain a white solid; the solid is then added with 2812g methanol to stir and dissolve, and 4921g isopropyl ether is added dropwise until the feed liquid is turbid. Maintain the internal temperature at 0-5 degrees, crystallize for 10 hours, filter with suction, and dry the filter cake at an external temperature of 40 degrees to obtain 694.4g white solid, yield: 93.1%.

[0052] As a result, the purity of the obtained product was 98.68% and the total yield was 79.9%.

[0053] Embodiment 3:

[0054] Step 1: Synthesis of H-Gly-His-OH

[0055] 5L three-necked flask, add 600g THF, 200g H-Gly-OH, add 830.1g triphosgene under ice bath, after adding, react at internal temperature of 25 degrees for 3h, concentrate THF, add the residue to 1.0kg methyl tert-butyl ether, filter and obtain yellow solid 1; 5L three-necked flask, add 1kg water, 392.71g L-histidine, adjust pH to 10.2 with 30% sodium hydroxide solution, control the temperature at 10-15℃, add solid 1, and control the pH of the feed liquid to 10.0-10.4 with sodium hydroxide solution. After adding, continue to react at external temperature of 25 degrees for 1h; adjust pH to 7.0, concentrate to remove water, add 2.6kg ethanol, stir and crystallize for 2h, filter and dry the filter cake in vacuo at 45 degrees to obtain 510.3g off-white solid, yield: 95.0%.

[0056] Step 2: Synthesis of Boc-Gly-His(Boc)-OH

[0057] In a 10L three-necked flask, add 2.55kg THF, 510.3g H-Gly-His-OH, and 1.15kg Boc anhydride, stir until dissolved, maintain the internal temperature at 5-10 degrees, dropwise add 535.35g triethylamine, after the dropwise addition is complete, react at an internal temperature of 45 degrees for 3h, and concentrate to dryness at 55 degrees to obtain 940.2g light yellow oil. Yield: 94.8%.

[0058] Step 3: Synthesis of Boc-Gly-His(Boc)-Lys(Boc)-OH

[0059] In a 10L three-necked flask, add 3700g THF, 340.16g Oxyma, 940.2g Boc-Gly-His(Boc)-OH, stir and dissolve, keep the internal temperature at 5-10 degrees, and drop the THF solution of DCC (493.87g DCC is dissolved in 1000g THF); after the drop is complete, react at an internal temperature of 25 degrees for 3 hours; add 1880g water, 533.4g H--Lys(Boc)-OH, and a mixed solution of 201.1g sodium bicarbonate and keep warm for 2 hours. After the reaction is completed by HPLC detection, filter, and concentrate the filtrate at an external temperature of 45 degrees. Adjust the pH to 2.0 and extract once with 2821g ethyl acetate. Wash the ethyl acetate phase with acid water at pH 2.0 three times, 1880g each time. Wash the washed ethyl acetate phase with 1880g saturated sodium chloride once, dry with 141g anhydrous magnesium sulfate, and filter. The filtrate was concentrated at an external temperature of 45 degrees to obtain a light yellow oil. 1880g of acetone was added to the bottle containing the oil, and the temperature was 60 degrees to stir and dissolve, and then the temperature was naturally cooled to room temperature to precipitate solids, which were filtered by suction and the filter cake was dried at an external temperature of 40 degrees to obtain 1.32kg of off-white solids with a yield of 95.0%.

[0060] Step 4: Synthesis of H-Gly-His-Lys-OH

[0061] 5L three-necked flask, add 1.452kg concentrated hydrochloric acid, 1.32kg Boc-Gly-His(Boc)-Lys(Boc)-OH, stir to dissolve, raise the internal temperature to 90 degrees and react for 6 hours. After the reaction is completed by HPLC detection, lower the internal temperature to 25℃, add 5940g THF to the reaction solution, grow crystals for 3 hours, and then filter to obtain a white solid; the solid is then added with 2376g methanol to stir and dissolve, and 3960g methyl tert-butyl ether is added dropwise until the feed liquid is turbid. Maintain the internal temperature at 0-5 degrees, crystallize for 10 hours, filter with suction, and dry the filter cake at an external temperature of 40 degrees to obtain 652.64g white solid, yield: 93.2%.

[0062] Results showed that the purity of the product was 98.49% and the total yield was 80.1%.

[0063] In order to more intuitively demonstrate the advantages of the liquid phase synthesis method of the present invention, the method of synthesizing oligopeptide-1 using the liquid phase of the present invention is compared with the method using the same process using equivalent replacement.

[0064] Comparative Example 1:

[0065] The synthesis method of oligopeptide-1 in this comparative example is to sequentially couple amino acids according to conventional synthesis methods, as follows:

[0066] Step 1: Synthesis of Boc-Gly-OH

[0067] In a 5L three-necked flask, add 500mL THF, 100g Gly, and 348.9g Boc anhydride, stir until dissolved, maintain the internal temperature at 5-10 degrees, add triethylamine dropwise, and after the addition is complete, react at an internal temperature of 45 degrees for 3 hours, and concentrate to dryness at 55 degrees to obtain 204g of light yellow oil. Yield: 87.4%.

[0068] Step 2: Synthesis of H-His-OMe

[0069] In a 5L three-necked flask, add 1L THF and 200g L-histidine, lower the temperature to 5°C, dropwise add 184g thionyl chloride, and react at 45°C for 3h, then concentrate to dryness at 55°C to obtain 210g of light yellow oil. Yield: 96.3%.

[0070] Step 3: Synthesis of Boc-Gly-His-OMe

[0071] In a 10L three-necked flask, add 1.5L THF, 204g Boc-Gly-OH, 206.86g H-His-OMe, 529.96g HBTU, stir and dissolve, keep the internal temperature at 5-10 degrees, and add 161.01g DIEA dropwise; after the dripping is completed, the internal temperature is 25 degrees and the reaction is about 2h; after the reaction is completed by HPLC detection, filter, and concentrate the filtrate at an external temperature of 45 degrees. Adjust the pH to 2.0 and extract once with 1L ethyl acetate. Wash the ethyl acetate phase with water at pH 2.0 three times, 300ml each time. Wash the ethyl acetate phase with water at pH 9.0 three times, 300ml each time. Wash the washed ethyl acetate phase with saturated sodium chloride once, dry with 50g anhydrous magnesium sulfate, and filter. The filtrate is concentrated at an external temperature of 45 degrees to obtain a light yellow oil. Add 1000 ml of acetone to the bottle containing the oil, stir at an external temperature of 60 degrees to dissolve, then cool to room temperature naturally to precipitate solid, filter it with suction, and dry the filter cake at an external temperature of 40 degrees to obtain an off-white solid.

[0072] Step 4: Synthesis of Boc-Gly-His-OH

[0073] 5L three-necked flask, add 1.5L methanol, 275.9g Boc-Gly-His-OMe, 53.21g lithium hydroxide monohydrate, stir to dissolve, raise to 45 degrees internal temperature and react for 3h. After the reaction is completed by HPLC detection, filter, and concentrate the filtrate at 45 degrees external temperature. Adjust the pH to 2.0 and extract once with 1L ethyl acetate. Wash the ethyl acetate phase with water at pH 2.0 three times, 300ml each time. Wash the ethyl acetate phase with saturated sodium chloride once, dry with 50g anhydrous magnesium sulfate, and filter. The filtrate is concentrated at 45 degrees external temperature until it disappears, and an off-white solid is obtained.

[0074] Step 5: Synthesis of Boc-Gly-His-Lys(Boc)-OH

[0075] In a 10L three-necked flask, add 1.0L THF, 138.14g Oxyma, 253g Boc-Gly-His-OH, stir and dissolve, keep the internal temperature at 5-10 degrees, and drop the THF solution of DCC (200.57g DCC is dissolved in 300ml THF); after the drop is complete, react at an internal temperature of 25 degrees for about 3 hours; add 1.3L water, 199.52g H--Lys(Boc)-OH, and 81.65g sodium bicarbonate. After the reaction is complete, filter and concentrate the filtrate at an external temperature of 45 degrees. Adjust the pH to 2.0 and extract once with 2L ethyl acetate. Wash the ethyl acetate phase with water at pH 2.0 three times, 600ml each time. Wash the washed ethyl acetate phase with saturated sodium chloride once, dry with 100g anhydrous magnesium sulfate, and filter. Concentrate the filtrate at an external temperature of 45 degrees to obtain a light yellow oil. Add 1000 ml of acetone to the bottle containing the oil, stir at an external temperature of 60 degrees to dissolve, then cool to room temperature naturally to precipitate solid, filter it with suction, and dry the filter cake at an external temperature of 40 degrees to obtain an off-white solid.

[0076] Step 6: Synthesis of H-Gly-His-Lys-OH

[0077] In a 5L three-necked flask, add 500mL of concentrated hydrochloric acid and 315.3g of Boc-Gly-His-Lys(Boc)-OH, stir until dissolved, then raise the internal temperature to 90 degrees and react for 6 hours. After the reaction is completed as detected by HPLC, lower the internal temperature to 25°C, add 2.5L of THF to the reaction solution, grow the crystal for 3 hours, and then filter to obtain a white solid; add 400ml of methanol to stir and dissolve the solid, and add 2L of ether until the liquid becomes turbid. Maintain the internal temperature at 0-5 degrees, crystallize for 10 hours, filter, and dry the filter cake at an external temperature of 40 degrees to obtain a white solid;

[0078] Comparative Example 2

[0079] If other protecting groups, such as Trt, are used in step 2, the reaction purity will be slightly lower and the by-products produced will be difficult to handle, so the BOC protecting group is selected.

[0080] The preparation method is the same as that in Example 1, except that the protecting group is Ttr, and the step 3 is to couple H--Lys(Trt)-OH, as follows:

[0081] Step 2: Synthesis of Trt-Gly-His(Trt)-OH

[0082] In a 10L three-necked flask, add 2.4L THF, 1.2L water, 536.6g GH, 1.69kg Trt-Cl, stir until dissolved, maintain the internal temperature at 5-10 degrees, add triethylamine dropwise, after the addition is complete, react at 45 degrees for 3h, add 6L water, adjust pH to 4.0, grow crystals for 2h, filter with suction, dry the filter cake at 50°C, and obtain 1.677kg white solid. Yield: 94.9%.

[0083] Step 3: Synthesis of Trt-Gly-His(Trt)-Lys(Trt)-OH

[0084] 10L three-necked flask, add 3L THF, 410.38g Oxyma, 1.677kg Trt-Gly-His(Trt)-OH, stir and dissolve, keep the internal temperature at 5-10 degrees, add DCC THF solution (595.82g DCC is dissolved in 1.5L THF); after dripping, react for about 3h at an internal temperature of 25 degrees; add 1.5L water, 591.5g H--Lys(Trt)-OH, and 242.27g sodium bicarbonate. After the reaction is completed by HPLC detection, add water to 10L. Adjust the pH to 4.0, precipitate solid, filter with suction, dry the filter cake at an external temperature of 40 degrees, and obtain 2.06kg off-white solid with a yield of 80.0%. The yield is low due to the steric hindrance of the protecting group Trt, solubility reasons and residual by-product triphenylmethanol.

[0085] Step 4: Synthesis of H-Gly-His-Lys-OH

[0086] In a 5L three-necked flask, add 1.2L concentrated hydrochloric acid, 915.56g TIPS, and 2.06kg Trt-Gly-His(Trt)-Lys(Trt)-OH, stir to dissolve, and then raise the internal temperature to 90 degrees to react for 6 hours. After the reaction is completed by HPLC detection, lower the internal temperature to 25°C, filter, and then add 6L THF to the filtrate, grow crystals for 3 hours, and then filter to obtain a white solid; the solid is then added with 3000ml methanol to stir and dissolve, and 6L ether is added dropwise until the feed liquid is turbid. Maintain the internal temperature at 0-5 degrees, crystallize for 10 hours, filter, and dry the filter cake at an external temperature of 40 degrees to obtain a white solid.

[0087] Comparative Example 3

[0088] If Oxyma in step 3 is replaced by HONp, etc., the yield is low.

[0089] The preparation method is the same as that in Example 1, except that Oxyma in step 3 is replaced by HONp; when used, the specific operation is as follows:

[0090] Step 3: Synthesis of Boc-Gly-His(Boc)-Lys(Boc)-OH

[0091] In a 10L three-necked flask, add 2.7L THF, 358.6g HONp, 886g Boc-Gly-His(Boc)-OH, stir and dissolve, keep the internal temperature at 5-10 degrees, add DCC THF solution dropwise (531.89g DCC is dissolved in 800ml THF); after the dripping is completed, react at an internal temperature of 25 degrees for about 3h; add 1.8L water, 529.11g H--Lys(Boc)-OH, and 216.72g sodium bicarbonate. After the reaction is completed by HPLC, filter, and concentrate the filtrate at an external temperature of 45 degrees. Adjust the pH to 2.0 and extract once with 3L ethyl acetate. Wash the ethyl acetate phase with water at pH 2.0 three times, 1000ml each time. Wash the washed ethyl acetate phase with saturated sodium chloride once, dry with 150g anhydrous magnesium sulfate, and filter. Concentrate the filtrate at an external temperature of 45 degrees to obtain a light yellow oil. Add 2000 ml of acetone to the bottle containing the oil, stir at an external temperature of 60 degrees to dissolve, then cool to room temperature naturally to precipitate solid, filter it with suction, and dry the filter cake at an external temperature of 40 degrees to obtain an off-white solid.

[0092] The data is as follows:

[0093] Table 1: Comparison of the purity and yield of the products prepared in different embodiments and comparative examples

[0094] purity Yield Example 1 98.77％ 80.3％ Example 2 98.68％ 79.9％ Example 3 98.49％ 80.1％ Comparative Example 1 98.46％ 37.9％ Comparative Example 2 97.18％ 62.18％ Comparative Example 3 98.24％ 62.03％

[0095] From the data analysis in Table 1, we can see that:

[0096] (1) Comparison between Example 1 and Example 1 shows that the synthesis method of oligopeptide-1 in Example 1 sequentially couples amino acids according to conventional synthesis methods. The purity of the obtained product is 98.46%, and the total yield is 37.9%, which is significantly low. The cycle is 94.5 hours, which is 21% longer than the 78 hours of the present invention. In addition, due to the long steps and large post-processing losses, the yield is low.

[0097] (2) Comparison between Comparative Example 2 and Example 1 shows that the preparation method is the same as Example 1, except that: Ttr is selected as the protecting group, and H--Lys(Trt)-OH is coupled in step 3. The reaction purity of Ttr is slightly low, and the by-products produced are difficult to handle, so BOC protecting group is selected. Due to the influence of the hydrolysis by-product Trt-TIS, the yield is slightly low. As a result, the purity of the obtained product is 97.18%, and the yield is 62.18%;

[0098] (3) Comparison between Comparative Example 2 and Example 1 shows that the difference from Example 1 is that Oxyma in step 3 is replaced by HONp; since HONp has good stability after binding and slow reaction, the yield is low. The purity of the obtained product is 98.24% and the yield is 62.03%.

[0099] In summary,

[0100] (1) The oligopeptide-1 prepared by the liquid phase synthesis method of the present invention reduces the production cycle, reduces the generation of waste liquid, and reduces the production cost;

[0101] (2) The present invention creatively adds two protecting groups in one step and removes all protecting groups in one step, thereby improving the purity of the intermediate product, reducing the purification pressure, and further improving the total yield, while shortening the production cycle and improving the reaction purity.

Claims

1. A method for synthesizing oligopeptide-1, characterized in that The steps include: Step 1: Synthesis of H-Gly-His-OH; Glycine and reaction solvent THF were added to the container, triphosgene was added under ice bath, the internal temperature was 25±2°C for 3h, the solvent was concentrated, the residue was added to methyl tert-butyl ether, and the mixture was filtered to obtain yellow solid 1; water and L-histidine were added to the container, the pH was adjusted to 10.2±0.2 with 30% sodium hydroxide solution, the temperature was controlled at 10-15°C, solid 1 was added, and the pH of the feed solution was controlled to 10.2±0.2 with sodium hydroxide solution. After the addition was completed, the external temperature was 25±2°C for further reaction for 1h; the pH was adjusted to 7.0-7.5, the water was concentrated, ethanol was added, stirred and crystallized for 2h, and then filtered, and the filter cake was vacuum dried at 45±2°C to obtain H-Gly-His-OH; Step 2: Synthesis of Boc-Gly-His(Boc)-OH; Add reaction solvent, H-Gly-His-OH and Boc anhydride to the container, stir until dissolved, maintain the internal temperature at 5-10 degrees, add triethylamine dropwise, and react at 45±2℃ for 3h, then concentrate to dryness at 55±2℃ to obtain Boc-Gly-His(Boc)-OH; Step 3: Synthesis of Boc-Gly-His(Boc)-Lys(Boc)-OH; Add THF, Oxyma, and Boc-Gly-His(Boc)-OH to a container, stir until dissolved, maintain the internal temperature at 5-10°C, and dropwise add a mixed solution of DCC; after the dropwise addition is complete, react at an internal temperature of 25±2°C for 3 hours; add a mixed solution of water, H--Lys(Boc)-OH, and sodium bicarbonate, and keep the temperature for 2 hours; after the reaction is complete, detect by HPLC, filter, and concentrate the filtrate at an external temperature of 45±2°C; Adjust the pH to 2.2±0.2, extract once with ethyl acetate; wash the ethyl acetate phase with acid water at pH 2.2±0.2 three times; wash the washed ethyl acetate phase with saturated brine once, dry with anhydrous magnesium sulfate, and filter; concentrate the filtrate at an external temperature of 45±2°C until no oil is obtained to obtain a light yellow oil; add acetone, stir at an external temperature of 60±2°C to dissolve, cool to room temperature naturally, precipitate solid, filter with suction, and dry the filter cake at an external temperature of 40±2°C to obtain Boc-Gly-His(Boc)-Lys(Boc)-OH; Step 4: Synthesis of H-Gly-His-Lys-OH; Add concentrated hydrochloric acid and Boc-Gly-His(Boc)-Lys(Boc)-OH to the container, stir to dissolve, then raise the internal temperature to 90±2°C and react for 6 hours; after the reaction is completed as detected by HPLC, lower the internal temperature to 25±2°C, add THF to the reaction solution, grow the crystal for 3 hours, and then filter to obtain a white solid; add methanol to the solid and stir to dissolve, add solvent dropwise until the feed liquid becomes turbid; maintain the internal temperature at 0-5 degrees, crystallize for 10 hours, filter with suction, and dry the filter cake at an external temperature of 40±2°C to obtain oligopeptide-1.

2. The method for synthesizing oligopeptide-1 according to claim 1, characterized in that In the step 1, the molar ratio of glycine: triphosgene: histidine is 1: (1.05-1.2): (0.95-1).

3. The method for synthesizing oligopeptide-1 according to claim 2, characterized in that In the step 1, the mass ratio of glycine: reaction solvent: methyl tert-butyl ether: water: ethanol is 1: (3-5): (5-6): 5: (13-15).

4. The method for synthesizing oligopeptide-1 according to claim 1, characterized in that In the step 2, the molar ratio of H-Gly-His-OH: Boc anhydride: triethylamine is 1: (2.2-2.4): (2.2-2.4).

5. The method for synthesizing oligopeptide-1 according to claim 4, characterized in that In the step 2, the mass ratio of H-Gly-His-OH to reaction solvent is 1:(5-6).

6. The method for synthesizing oligopeptide-1 according to claim 1, characterized in that In the step 3, the molar ratio of Boc-Gly-His(Boc)-OH:Oxyma:DCC:H--Lys(Boc)-OH:sodium bicarbonate is 1:(1.05-1.2):(1.05-1.2):(0.95-1.0):(1.05-1.2).

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